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Abstract Methane (CH4) emissions from natural systems are rising in a concerning manner with an incomplete understanding of its drivers. Recently, chemical stressors such as antibiotics have been suggested as a thus far overlooked factor increasing methanogenesis in freshwaters. Since usage and toxicological impact of antibiotics could increase in a warming climate, we assessed the temperature-dependence of antibiotic effects on methanogenesis. In this light, we conducted anaerobic incubations with freshwater sediment at 10, 15, and 20 °C in presence of a mixture of five antibiotics at field-relevant concentrations. Weekly measurements of CH4 showed a strong temperature dependence of antibiotic effects by changing effect sizes, directions and dynamics. While antibiotics reduced CH4 production at 10 °C, methanogenesis was elevated at 15 °C with the most pronounced increase occurring at 20 °C. Furthermore, antibiotics changed the prokaryotic assemblage at all temperatures and effect patterns of CH4 producing Methanomicrobia strongly followed the patterns observed for methanogenesis. While analyses of compound-specific stable isotopes and the metatranscriptome suggest the acetoclastic pathway as most relevant, linking prokaryotic structure to function remains one of the most significant research challenges. Nevertheless, the evidence provided by this study suggests a positive relationship between temperature and the stimulating effects of antibiotics on CH4 production.

Background: Theta-gamma transcranial alternating current stimulation (tACS) was recently found to enhance thumb acceleration in young, healthy participants, suggesting a potential role in facilitating motor skill acquisition. Given the relevance of motor skill acquisition in stroke rehabilitation, theta-gamma tACS may hold potential for treating stroke survivors. Objective: We aimed to examine the effects of theta-gamma tACS on motor skill acquisition in young, healthy participants and stroke survivors. Methods: In a pre-registered, double-blind, randomized, sham-controlled study, 78 young, healthy participants received either theta-gamma peak-coupled (TGP) tACS, theta-gamma trough-coupled (TGT) tACS or sham stimulation. 20 individuals with a chronic stroke received either TGP or sham. TACS was applied over motor cortical areas while participants performed an acceleration-dependent thumb movement task. Stroke survivors were characterized using standardized testing, with a subgroup receiving additional structural brain imaging. Results: Neither TGP nor TGT tACS significantly modified general motor skill acquisition in the young, healthy cohort. In contrast, in the stroke cohort, TGP diminished motor skill acquisition compared to sham. Exploratory analyses revealed that, independent of general motor skill acquisition, healthy participants receiving TGP or TGT exhibited greater peak thumb acceleration than those receiving sham. Conclusion: Although theta-gamma tACS increased thumb acceleration in young, healthy participants, consistent with previous reports, it did not enhance overall motor skill acquisition in a more complex motor task. Furthermore, it even had detrimental effects on motor skill acquisition in stroke survivors.

Abstract Background Obstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. As studies addressing limb muscles are rare, the reasons for the lower exercise capacity are unknown. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles. Methods Mice were kept under 6 weeks of CIH (alternating 7% and 21% O2 fractions every 30 s, 8 h/day, 5 days/week) compared to normoxia (NOX). Analyses included neuromuscular junctions (NMJ) postsynaptic morphology and integrity, fiber cross-sectional area (CSA) and composition (ATPase), mitochondrial ultrastructure (transmission-electron-microscopy), and relevant transcripts (RT-qPCR). Besides wildtype (WT), we included inducible nitric oxide synthase knockout mice (iNOS−/−) to evaluate whether iNOS is protective or risk-mediating. Results In WT soleus muscle, CIH vs. NOX reduced NMJ size (− 37.0%, p < 0.001) and length (− 25.0%, p < 0.05) together with fiber CSA of type IIa fibers (− 14%, p < 0.05) and increased centronucleated fiber fraction (p < 0.001). Moreover, CIH vs. NOX increased the fraction of damaged mitochondria (1.8-fold, p < 0.001). Compared to WT, iNOS−/− similarly decreased NMJ area and length with NOX (− 55%, p < 0.001 and − 33%, p < 0.05, respectively) or with CIH (− 37%, p < 0.05 and − 29%, p < 0.05), however, prompted no fiber atrophy. Moreover, increased fractions of damaged (2.1-fold, p < 0.001) or swollen (> 6-fold, p < 0.001) mitochondria were observed with iNOS−/− vs. WT under NOX and similarly under CIH. Both, CIH- and iNOS−/− massively upregulated suppressor-of-cytokine-signaling-3 (SOCS3) > 10-fold without changes in IL6 mRNA expression. Furthermore, inflammatory markers like CD68 (macrophages) and IL1β were significantly lower in CIH vs. NOX. None of these morphological alterations with CIH- or iNOS−/− were detected in the gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH. Conclusion CIH leads to NMJ and mitochondrial damage associated with fiber atrophy/centronucleation selectively in slow-twitch muscle of WT. This effect is largely mimicked by iNOS−/− at NOX (except for atrophy). Both conditions involve massive SOCS3 upregulation likely through denervation without Il6 upregulation but accompanied by a decrease of macrophage density especially next to denervated endplates. In the absence of muscular iNOS expression in WT, this damage may arise from extramuscular, e.g., motoneuronal iNOS deficiency (through CIH or knockout) awaiting functional evaluation.

Fungicides pose a risk for crustacean leaf shredders serving as key-stone species for leaf litter breakdown in detritus-based stream ecosystems. However, little is known about the impact of strobilurin fungicides on shredders, even though they are presumed to be the most hazardous fungicide class for aquafauna. Therefore, we assessed the impact of the strobilurin azoxystrobin (AZO) on the survival, energy processing (leaf consumption and feces production), somatic growth (growth rate and molting activity), and energy reserves (neutral lipid fatty and amino acids) of the amphipod crustacean Gammarus fossarum via waterborne exposure and food quality-mediated (through the impact of leaf colonizing aquatic microorganisms) and thus indirect effects using 2 × 2-factorial experiments over 24 days. In a first bioassay with 30 µg AZO/L, waterborne exposure substantially reduced survival, energy processing and affected molting activity of gammarids, while no effects were observed via the dietary pathway. Furthermore, a negative growth rate (indicating a body mass loss in gammarids) was induced by waterborne exposure, which cannot be explained by a loss in neutral lipid fatty and amino acids. These energy reserves were increased indicating a disruption of the energy metabolism in G. fossarum caused by AZO. Contrary to the first bioassay, no waterborne AZO effects were observed during a second experiment with 15 µg AZO/L. However, an altered energy processing was determined in gammarids fed with leaves microbially colonized in the presence of AZO, which was probably caused by fungicide-induced effects on the microbial decomposition efficiency ultimately resulting in a lower food quality. The results of the present study show that diet-related strobilurin effects can occur at concentrations below those inducing waterborne toxicity. However, the latter seems to be more relevant at higher fungicide concentrations.

Objective: Motor recovery after stroke shows a high inter-subject variability. The brain's potential to form new connections determines individual levels of recovery of motor function. Most of our daily activities require visuomotor integration, which engages parietal areas. Compared to the frontal motor system, less is known about the parietal motor system's reconfiguration related to stroke recovery. Here, we tested if functional connectivity among parietal and frontal motor areas undergoes plastic changes after stroke and assessed the behavioral relevance for motor function after stroke. Methods: We investigated stroke lesion-induced changes in functional connectivity by measuring high-density electroencephalography (EEG) and assessing task-related changes in coherence during a visually guided grip task with the paretic hand in 30 chronic stroke patients with variable motor deficits and 19 healthy control subjects. Quantitative changes in task-related coherence in sensorimotor rhythms were compared to the residual motor deficit. Results: Parietofrontal coupling was significantly stronger in patients compared to controls. Whereas motor network coupling generally increased during the task in both groups, the task-related coherence between the parietal and primary motor cortex in the stroke lesioned hemisphere showed increased connectivity across a broad range of sensorimotor rhythms. Particularly the parietofrontal task-induced coupling pattern was significantly and positively related to residual impairment in the Nine-Hole Peg Test performance and grip force. Interpretation: These results demonstrate that parietofrontal motor system integration during visually guided movements is stronger in the stroke-lesioned brain. The correlation with the residual motor deficit could either indicate an unspecific marker of motor network damage or it might indicate that upregulated parietofrontal connectivity has some impact on post-stroke motor function. Keywords: EEG, Coherence, Motor recovery, Stroke, Parietal lobe

New SARS-CoV-2 lineages continue to evolve and may exhibit new characteristics regarding host cell entry efficiency and potential for antibody evasion. Here, employing pseudotyped particles, we compared the host cell entry efficiency, ACE2 receptor usage, and sensitivity to antibody-mediated neutralization of four emerging SARS-CoV-2 lineages, KP.2, KP.2.3, KP.3, and LB.1. The XBB.1.5 and JN.1 lineages served as controls. Our findings reveal that KP.2, KP.2.3, KP.3, and LB.1 lineages enter host cells efficiently and in an ACE2-dependent manner, and that KP.3 is more adept at entering Calu-3 lung cells than JN.1. However, the variants differed in their capacity to employ ACE2 orthologues from animal species for entry, suggesting differences in ACE2 interactions. Moreover, we demonstrate that only two out of seven therapeutic monoclonal antibody (mAbs) in preclinical development retain robust neutralizing activity against the emerging JN.1 sublineages tested, while three mAbs displayed strongly reduced neutralizing activity and two mAbs lacked neutralizing activity against any of the lineages tested. Furthermore, our results show that KP.2, KP.2.3, KP.3, and LB.1 lineages evade neutralization by antibodies induced by infection or vaccination with greater efficiency than JN.1, particularly in individuals without hybrid immunity. This study indicates that KP.2, KP.2.3, KP.3, and LB.1 differ in ACE2 interactions and the efficiency of lung cell entry and suggest that evasion of neutralizing antibodies drove the emergence of these variants.

The COVID-19 pandemic, caused by SARS-CoV-2, demonstrated that zoonotic transmission of animal sarbecoviruses threatens human health but the determinants of transmission are incompletely understood. Here, we show that most spike (S) proteins of horseshoe bat and Malayan pangolin sarbecoviruses employ ACE2 for entry, with human and raccoon dog ACE2 exhibiting broad receptor activity. The insertion of a multibasic cleavage site into the S proteins increased entry into human lung cells driven by most S proteins tested, suggesting that acquisition of a multibasic cleavage site might increase infectivity of diverse animal sarbecoviruses for the human respiratory tract. In contrast, two bat sarbecovirus S proteins drove cell entry in an ACE2-independent, trypsin-dependent fashion and several ACE2-dependent S proteins could switch to the ACE2-independent entry pathway when exposed to trypsin. Several TMPRSS2-related cellular proteases but not the insertion of a multibasic cleavage site into the S protein allowed for ACE2-independent entry in the absence of trypsin and may support viral spread in the respiratory tract. Finally, the pan-sarbecovirus antibody S2H97 enhanced cell entry driven by two S proteins and this effect was reversed by trypsin while trypsin protected entry driven by a third S protein from neutralization by S2H97. Similarly, plasma from quadruple vaccinated individuals neutralized entry driven by all S proteins studied, and availability of the ACE2-independent, trypsin-dependent pathway reduced neutralization sensitivity. In sum, our study reports a pathway for entry into human cells that is ACE2-independent, can be supported by TMPRSS2-related proteases and may be associated with antibody evasion.

BackgroundThe treatment of head and neck tumors remains a challenge due to their reduced radiosensitivity. Small molecule kinase inhibitors (smKI) that inhibit the DNA damage response, may increase the radiosensitivity of tumor cells. However, little is known about how the immunophenotype of the tumor cells is modulated thereby. Therefore, we investigated whether the combination of ATM or ATR inhibitors with hypo-fractionated radiotherapy (RT) has a different impact on the expression of immune checkpoint markers (extrinsic), the release of cytokines or the transcriptome (intrinsic) of head and neck squamous cell carcinoma (HNSCC) cells.MethodsThe toxic and immunogenic effects of the smKI AZD0156 (ATMi) and VE-822 (ATRi) in combination with a hypo-fractionated scheme of 2x5Gy RT on HPV-negative (HSC4, Cal-33) and HPV-positive (UM-SCC-47, UD-SCC-2) HNSCC cell lines were analyzed as follows: cell death (necrosis, apoptosis; detected by AnxV/PI), expression of immunostimulatory (ICOS-L, OX40-L, TNFSFR9, CD70) and immunosuppressive (PD-L1, PD-L2, HVEM) checkpoint marker using flow cytometry; the release of cytokines using multiplex ELISA and the gene expression of Cal-33 on mRNA level 48 h post-RT.ResultsCell death was mainly induced by the combination of RT with both inhibitors, but stronger with ATRi. Further, the immune phenotype of cancer cells, not dying from combination therapy itself, is altered predominantly by RT+ATRi in an immune-stimulatory manner by the up-regulation of ICOS-L. However, the analysis of secreted cytokines after treatment of HNSCC cell lines revealed an ambivalent influence of both inhibitors, as we observed the intensified secretion of IL-6 and IL-8 after RT+ATRi. These findings were confirmed by RNAseq analysis and further the stronger immune-suppressive character of RT+ATMi was enlightened. We detected the down-regulation of a central protein of cytoplasmatic sensing pathways of nucleic acids, RIG-1, and found one immune-suppressive target, EDIL3, strongly up-regulated by RT+ATMi.ConclusionIndependent of a restrictive toxicity, the combination of RT + either ATMi or ATRi leads to comprehensive and immune-modulating alterations in HNSCC. This includes pro-inflammatory signaling induced by RT + ATRi but also anti-inflammatory signals. These findings were confirmed by RNAseq analysis, which further highlighted the immune-suppressive nature of RT + ATMi.

Abstract Immune-mediated inflammatory diseases (IMIDs) are typically characterised by relapsing and remitting flares of inflammation. However, the unpredictability of disease flares impedes their study. Addressing this critical knowledge gap, we use the experimental medicine approach of immunomodulatory drug withdrawal in rheumatoid arthritis (RA) remission to synchronise flare processes allowing detailed characterisation. Exploratory mass cytometry analyses reveal three circulating cellular subsets heralding the onset of arthritis flare – CD45RO+PD1hi CD4+ and CD8+ T cells, and CD27+CD86+CD21- B cells – further characterised by single-cell sequencing. Distinct lymphocyte subsets including cytotoxic and exhausted CD4+ memory T cells, memory CD8+CXCR5+ T cells, and IGHA1+ plasma cells are primed for activation in flare patients. Regulatory memory CD4+ T cells (Treg cells) increase at flare onset, but with dysfunctional regulatory marker expression compared to drug-free remission. Significant clonal expansion is observed in T cells, but not B cells, after drug cessation; this is widespread throughout memory CD8+ T cell subsets but limited to the granzyme-expressing cytotoxic subset within CD4+ memory T cells. Based on our observations, we suggest a model of immune dysregulation for understanding RA flare, with potential for further translational research towards novel avenues for its treatment and prevention.

Abstract Background Clinical Practice Guidelines for Gulf War Illness (GWI) recommend integrative health approaches such as yoga for relief from symptoms, yet little is known about the long-term efficacy of yoga in reducing symptoms of GWI. Here, we evaluated the long-term efficacy of yoga and cognitive-behavioral therapy (CBT) chronic pain treatment in a randomized controlled trial (RCT) of 75 Veterans (57 men, 42–71 ± 7.1 years of age) with Gulf War Illness (GWI). Methods Participants received either 10 weeks of yoga or 10 weeks of CBT for chronic pain. The primary outcome measures were pain severity, and pain interference (Brief Pain Inventory-Short Form). The secondary outcome measures were fatigue, as indicated by a measure of functional exercise capacity (6-Minute Walk Test), depression, autonomic symptom severity, and quality of life. Piecewise linear mixed models were used to examine study hypotheses. Results Compared to the CBT group, yoga was associated with greater reductions in pain severity during the 6-month follow-up period (group × time interaction: b = 0.036, se = 0.014, p = .011). Although we did not find between-group differences in the other primary or secondary outcome measures during follow-up (p’s > 0.05), exploratory analyses revealed within-group improvements in pain interference, total pain (an experimental outcome variable which combines pain severity and interference), and fatigue in the yoga group (p’s

Summary: Glucose uptake increases during B cell activation and antibody-secreting cell (ASC) differentiation, but conflicting findings prevent a clear metabolic profile at different stages of B cell activation. Deletion of the glucose transporter type 1 (GLUT1) gene in mature B cells (GLUT1-cKO) results in normal B cell development, but it reduces germinal center B cells and ASCs. GLUT1-cKO mice show decreased antigen-specific antibody titers after vaccination. In vitro, GLUT1-deficient B cells show impaired activation, whereas established plasmablasts abolish glycolysis, relying on mitochondrial activity and fatty acids. Transcriptomics and metabolomics reveal an altered anaplerotic balance in GLUT1-deficient ASCs. Despite attempts to compensate for glucose deprivation by increasing mitochondrial mass and gene expression associated with glycolysis, the tricarboxylic acid cycle, and hexosamine synthesis, GLUT1-deficient ASCs lack the metabolites for energy production and mitochondrial respiration, limiting protein synthesis. We identify GLUT1 as a critical metabolic player defining the germinal center response and humoral immunity.

Transmissibility and immune evasion of the recently emerged, highly mutated SARS-CoV-2 BA.2.87.1 are unknown. Here, we report that BA.2.87.1 efficiently enters human cells but is more sensitive to antibody-mediated neutralization than the currently dominating JN.1 variant. Acquisition of adaptive mutations might thus be needed for efficient spread in the population.

Summary: Background: SARS-CoV-2 specific antibody-secreting plasma cells (PC) mediating specific humoral immunity have been identified in the human bone marrow (BM) after COVID-19 or vaccination against SARS-CoV-2. However, it remained unclear whether or not they acquire phenotypes of human memory plasma cells. Methods: SARS-CoV-2-specific human bone marrow plasma cells (BMPC) were characterised by tetramer-based, antigen-specific flow cytometry and FluoroSpot assay. Findings: SARS-CoV-2 spike-S1-specific PC were detectable in all tested BM samples of previously vaccinated individuals, representing 0.22% of total BMPC. The majority of SARS-CoV-2-specific BMPC expressed IgG and their specificity for the spike S1 protein indicated emergence from a systemic vaccination response. Of note, one-fifth of SARS-CoV-2-specific BMPC showed the phenotype of memory plasma cells, i.e., downregulated CD19 and present or absent CD45 expression. Interpretation: Our data indicate the establishment of phenotypically diverse SARS-CoV-2-specific PC in the human BM after basic mRNA immunization, including the formation of memory phenotypes. These results suggest the induction of durable humoral immunity after basic mRNA vaccination against SARS-CoV-2. Funding: The study was supported by funding by the DFG grants TRR130 TP24, ME 3644/8-1, and the Berlin Senate. SR received funding from DFG SFB-1444 C01 Central Service Project.

We have previously shown that the microRNA (miRNA) processor complex consisting of the RNAse Drosha and the DiGeorge Critical Region (DGCR) 8 protein is essential for B cell maturation. To determine whether miRNA processing is required to initiate T cell-mediated antibody responses, we deleted DGCR8 in maturing B2 cells by crossing a mouse with loxP-flanked DGCR8 alleles with a CD23-Cre mouse. As expected, non-immunized mice showed reduced numbers of mature B2 cells and IgG-secreting cells and diminished serum IgG titers. In accordance, germinal centers and antigen-specific IgG-secreting cells were absent in mice immunized with T-dependent antigens. Therefore, DGCR8 is required to mount an efficient T-dependent antibody response. However, DGCR8 deletion in B1 cells was incomplete, resulting in unaltered B1 cell numbers and normal IgM and IgA titers in DGCR8-knock-out mice. Therefore, this mouse model could be used to analyze B1 responses in the absence of functional B2 cells.

Understanding leaf generation dynamics, their seasonal changes, and their responses to nitrogen fertilization (NF) is key to improving pasture utilization efficiency. The objectives of this research were to determine structural and morphogenetic variables underlying changes in herbage mass on a set of Paspalum notatum genotypes. Ten P. notatum genotypes were evaluated in experimental plots following a completely randomized block design under a split-plot arrangement for two N-rates during four periods. Increased herbage mass (HM) after N-fertilization was explained by a higher tiller density (TD) (41.8%) and tiller weight (TW) (22.1%). The increment of TW after NF was due to the increase in leaf blade length (LBL) and width (LBW). During the flowering season, NF increases the reproductive tiller density by 262.5%. Seasonal variation in HM was mainly explained by changes in LBL that modified TW. Morphogenetic traits differed between genotypes of different growth habits; therefore, different management practices are suggested. The average increase in leaf elongation rate in response to NF was about 36.7%, generating longer leaves despite reductions in leaf elongation time (LET). The depletion in LBL and consequently in TW and HM during the autumn was attributed to the reduction in LET.

ABSTRACT: Objectives: There have been efforts to promote timely antimicrobial administration for patients with sepsis, but the importance for other infections is uncertain. This study analysed whether time to first antimicrobial dose (TFAD) in patients with lower limb cellulitis influenced outcome measures such as acute length of stay (LOS) in hospital and 30-day hospital readmission rates for cellulitis. Methods: Medical records of patients admitted with lower limb cellulitis or erysipelas over a 15-month period (1 May 2019 to 30 November 2019 and 1 March 2020 to 31 October 2020) were reviewed. Patients requiring intensive care unit (ICU) admission were excluded. The TFAD was the difference (in minutes) between the emergency department triage time and the time that the antimicrobial was first recorded as administered. Analysis included log-transformed linear regression (for LOS) and logistic regression (for 30-day readmission with cellulitis), controlling for confounders where possible. Results: The study included 282 patients with lower limb cellulitis. The median TFAD was 177 min (interquartile range, 98–290 min). Linear regression suggested a weak association between TFAD and LOS (P = 0.05; adjusted R2 = 0.01), which was non-significant after adjusting for confounders (P = 0.18). There were too few patients readmitted within 30 days with cellulitis for meaningful analysis. Conclusion: After controlling for confounders, no association between increased TFAD and increased acute LOS was identified for patients with lower limb cellulitis who did not require ICU admission (i.e. without septic shock). Conclusions could not be made for 30-day readmission rates for cellulitis.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While TALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALL is therefore urgently needed, together with precision medicine tools allowing the testing of efficacy in patient samples. Furthermore, leukemic cell heterogeneity requires drug response assessment at the single-cell level. Here we used single-cell mass cytometry to study signal transduction pathways such as JAK-STAT, PI3K-AKT-mTOR and MEK-ERK in 16 diagnostic and five relapsed T-ALL primary samples, and investigated the in vitro response of cells to Interleukin-7 (IL-7) and the inhibitor BEZ-235. T-ALL cells showed upregulated activity of the PI3K-AKT-mTOR and MEK-ERK pathways and increased expression of proliferation and translation markers. We found that perturbation induced by the ex vivo administration of either IL-7 or BEZ-235 reveals a high degree of exclusivity with respect to the phospho-protein responsiveness to these agents. Notably, these response signatures were maintained from diagnosis to relapse in individual patients. In conclusion, we demonstrated the power of mass cytometry single-cell profiling of signal transduction pathways in T-ALL. Taking advantage of this advanced approach, we were able to identify distinct clusters with different responsiveness to IL-7 and BEZ-235 that can persist at relapse. Collectively our observations can contribute to a better understanding of the complex signaling network governing T-ALL behavior and its correlation with influence on the response to therapy.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitates viral entry into host cells and is the key target for neutralizing antibodies. The SARS-CoV-2 lineage B.1.620 carries fifteen mutations in the S protein and is spread in Africa, the US and Europe, while lineage R.1 harbors four mutations in S and infections were observed in several countries, particularly Japan and the US. However, the impact of the mutations in B.1.620 and R.1 S proteins on antibody-mediated neutralization and host cell entry are largely unknown. Here, we report that these mutations are compatible with robust ACE2 binding and entry into cell lines, and they markedly reduce neutralization by vaccine-induced antibodies. Our results reveal evasion of neutralizing antibodies by B.1.620 and R.1, which might have contributed to the spread of these lineages.

Summary: Plasma cells secreting affinity-matured antibodies develop in germinal centers (GCs), where B cells migrate persistently and directionally over defined periods of time. How modes of GC B cell migration influence plasma cell development remained unclear. Through genetic deletion of the F-actin bundling protein Swiprosin-1/EF-hand domain family member 2 (EFhd2) and by two-photon microscopy, we show that EFhd2 restrains B cell speed in GCs and hapten-specific plasma cell output. Modeling the GC reaction reveals that increasing GC B cell speed promotes plasma cell generation. Lack of EFhd2 also reduces contacts of GC B cells with follicular dendritic cells in vivo. Computational modeling uncovers that both GC output and antibody affinity depend quantitatively on contacts of GC B cells with follicular dendritic cells when B cells migrate more persistently. Collectively, our data explain how GC B cells integrate speed and persistence of cell migration with B cell receptor affinity.

In highly aggressive malignancies like pancreatic cancer (PC), patient-derived tumor models can serve as disease-relevant models to understand disease-related biology as well as to guide clinical decision-making. In this study, we describe a two-step protocol allowing systematic establishment of patient-derived primary cultures from PC patient tumors. Initial xenotransplantation of surgically resected patient tumors (n = 134) into immunodeficient mice allows for efficient in vivo expansion of vital tumor cells and successful tumor expansion in 38% of patient tumors (51/134). Expansion xenografts closely recapitulate the histoarchitecture of their matching patients’ primary tumors. Digestion of xenograft tumors and subsequent in vitro cultivation resulted in the successful generation of semi-adherent PC cultures of pure epithelial cell origin in 43.1% of the cases. The established primary cultures include diverse pathological types of PC: Pancreatic ductal adenocarcinoma (86.3%, 19/22), adenosquamous carcinoma (9.1%, 2/22) and ductal adenocarcinoma with oncocytic IPMN (4.5%, 1/22). We here provide a protocol to establish quality-controlled PC patient-derived primary cell cultures from heterogeneous PC patient tumors. In vitro preclinical models provide the basis for the identification and preclinical assessment of novel therapeutic opportunities targeting pancreatic cancer.

OBJECTIVES: Refugees and immigrants from developing countries settling in industrialised countries have a high prevalence of Helicobacter pylori (H. pylori). Screening these groups for H. pylori and use of eradication therapy to reduce the future burden of gastric cancer and peptic ulcer disease is not currently recommended in most countries. We investigated whether a screening and eradication approach would be cost effective in high prevalence populations. METHODS: Nine different screening and follow-up strategies for asymptomatic immigrants from high H. pylori prevalence areas were compared with the current approach of no screening. Cost effectiveness comparisons assumed population prevalence's of H. pylori of 25%, 50% or 75%. The main outcome measure was the net cost for each cancer prevented for each strategy. Total costs of each strategy and net costs including savings from reductions in ulcers and gastric cancer were also calculated. RESULTS: Stool antigen testing with repeat testing after treatment was the most cost effective approach relative to others, for each prevalence value. The net cost per cancer prevented with this strategy was US$111,800 (assuming 75% prevalence), $132,300 (50%) and $193,900 (25%). A test and treat strategy using stool antigen remained relatively cost effective, even when the prevalence was 25%. CONCLUSIONS: H. pylori screening and eradication can be an effective strategy for reducing rates of gastric cancer and peptic ulcers in high prevalence populations and our data suggest that use of stool antigen testing is the most cost effective approach.

Observations of the mollusc-hunting cone snail Conus textile during feeding reveal that prey are often stung multiple times in succession. While studies on the venom peptides injected by fish-hunting cone snails have become common, these approaches have not been widely applied to the analysis of the injected venoms from mollusc-hunters. We have successfully obtained multiple injected venom samples from C. textile individuals, allowing us to investigate venom compositional variation during prey capture. Our studies indicate that C. textile individuals alter the composition of prey-injected venom peptides during single feeding events. The qualitative results obtained by MALDI-ToF mass spectrometry are mirrored by quantitative changes in venom composition observed by reverse-phase high performance liquid chromatography. While it is unclear why mollusc-hunting cone snails inject prey multiple times prior to engulfment, our study establishes for the first time a link between this behavior and compositional changes of the venom during prey capture. Changes in venom composition during hunting may represent a multi-step strategy utilized by these venomous animals to slow and incapacitate prey prior to engulfment.